Biosensor, method of manufacturing sensing unit thereof, and measuring system

ABSTRACT

A biosensor, a method of fabricating the sensing unit for the biosensor, and a measuring system comprising the biosensor. The biosensor has an extended gate field effect transistor (EGFET) structure and comprises a metal oxide semiconductor field effect transistor (MOSFET) on a semiconductor substrate, a sensing unit comprising a substrate, a silicon dioxide layer on the substrate, a tin oxide layer on the silicon dioxide layer, and a urease layer immobilized on the tin oxide layer, and a conductive wire connecting the MOSFET and the sensing unit.

BACKGROUND

[0001] The invention relates to a biosensor, and in particular to abiosensor having an extended gate field effect transistor (EGFET)structure with a urea layer immobilized on a tin oxide layer.

[0002] The first ion sensitive field effect transistor (ISFET) wasfabricated by P. Bergveld in 1970. The ISFET unlike the MOSFET has nometal gate electrode. Silicon dioxide (SiO₂) was first used as a pHsensitive membrane for the ISFET. Subsequently, Al₂O₃, Si₃N₄, Ta₂O₅, andSnO₂ were used as a pH sensitive membrane due to their higher pHresponse.

[0003] The earliest suggestion of the concept of an enzyme modified FET(EnFET) sensor device appears to be that of Janata and Moss. Theydescribed a penicillin responsive sensor which comprises a matchedpH-responsive ISFET pair, one device having an overlying, active gatefilm of the cross-linked albumin-penicillinase, and the other having areference gate membrane of only cross-linked albumin. When penicillinwas present in the analysis, the penicillinase present in the activegate material catalyzed the hydrolysis of penicillin to penicilloicacid, which released protons and created a local decrease in pH, whereasthe reference gate remained unaffected. Several applications for theEnFET, such as the glucose, urea, acetylcholine, and alcohol exist.

[0004] A number of patents relating to ISFETs are listed hereinafter.

[0005] U.S. Pat. No. 6,218,208 discloses a sensitive material tin oxide(SnO₂) obtained by thermal evaporation or by R.F. reactive sputtering,used as a high pH-sensitive material for a multi-structure ISFET.

[0006] U.S. Pat. No. 5,602,467 discloses a circuit layout for measuringion concentrations in solutions using ISFET. The circuit layout makes itpossible to represent the threshold voltage difference of the two ISFETsdirectly and independently of technological tolerances, operationallycaused parameter fluctuations, and ambient influences.

[0007] U.S. Pat. No. 5,387,328 discloses a biosensor employing an ISFETcomprising a source and a drain formed in a substrate, an ion sensitivegate placed between the source and drain, an ion sensitive film formedon the ion sensing gate, an immobilized enzyme membrane defined on theion sensitive film and, a Pt electrode formed on the ion sensitive film.The sensor has a Pt electrode capable of sensing all biologicalsubstances that generate H₂O₂ in the enzyme reaction, whereby areachieved the high sensitivity and rapid reaction time.

[0008] U.S. Pat. No. 5,350,701 discloses a process for producing asurface gate comprising a selective membrane for an integrated chemicalsensor comprising a field effect transistor, and the integrated chemicalsensor thus produced, wherein the surface gate is particularly sensitiveto alkaline-earth species, and more particularly, sensitive to thecalcium ion. The process comprises forming grafts on the surface gate,and making the grafts operative utilizing phosphonate based,ion-sensitive molecules.

[0009] U.S. Pat. No. 5,309,085 discloses a measuring circuit with abiosensor utilizing ion sensitive field effect transistors integratedinto one chip. The measuring circuit comprises two ion sensitive FETinput devices composed of an enzyme FET having an enzyme sensitivemembrane on the gate and a reference FET, and a differential amplifierfor amplifying the outputs of the enzyme FET and the reference FET.

[0010] A variety of materials are known to be capable of serving as thesensing film of an ISFETs, such as, Al₂O₃, Si₃N₄, a-WO₃, a-C:H, anda-Si:H, etc. The manufacture of sensing films is typically accomplishedby deposition, such as, sputtering or plasma enhanced chemical vapordeposition (PECVD), therefore, the cost is relatively high and the timerequired for thin film fabrication is long.

[0011] Thus, an easily fabricated, low cost ISFET and the sensing filmthereof, eliminating packing problems, are desirable.

SUMMARY

[0012] Accordingly, the biosensor according to the invention, having anextended gate field effect transistor structure, comprises a metal oxidesemiconductor field effect transistor, a sensing unit, and a conductivewire. The metal oxide semiconductor field effect transistor is formed ona semiconductor substrate. The sensing unit comprises a substrate, asilicon dioxide layer on the substrate, a tin oxide layer on the silicondioxide layer, and a urease layer immobilized on the tin oxide layer.The conductive wire connects the MOSFET and the sensing unit.

[0013] The method of manufacturing a sensing unit according to anembodiment of the invention comprises the steps of providing aconductive substrate; forming a silicon dioxide layer on the conductivesubstrate; forming a tin oxide layer on the silicon dioxide layer;electrically connecting the conductive substrate with a conductive wire;forming an insulating layer on the surface of the sensing unit andexposing part of the tin oxide layer and part of the conductive wire;and immobilizing a urease layer on the exposed part of tin oxide layerby gel entrapment.

[0014] The measuring system according to an embodiment of the inventioncomprises a biosensor as described above; a reference electrode forsupplying a stable voltage; an instrumentation amplifier having twoinputs and one output, wherein the two inputs are connected to thebiosensor and the reference electrode, respectively; a high-resistancemultimeter connected with the output of the instrumentation amplifier;and a computer connected with the high-resistance multimeter through acommunication interface card to store, process, or analyze data.

[0015] The biosensor according to an embodiment of the invention has theadvantages of short response time, simple packing, easy manufacture, andlow cost. When compared with conventional FET, this biosensor is morestable, provides better current-leakage protection, and highersensitivity. In one embodiment of the invention, the biosensor may havea sensitivity of 58 mV/pH and better linearity in a solution having a pHvalue in the range of 1 to 9, and may be used as a disposable sensingstructure.

[0016] In the method according to embodiments of the invention, a gelentrapment is performed by immobilization of an enzyme with aphotosensitive polymer on an ion sensitive film, thus the ISFET isfavorably used in biosensors. In addition, a commercializedSnO₂/SiO₂/glass can be directly used to manufacture the enzyme sensingfilm, thus manufacturing is simplified and cost is reduced.

[0017] In embodiments of the invention, the sensing structure isdisposable, thus eliminating problems of enzyme loss due to long-termuse and cross contamination.

[0018] A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments of the invention can be more fully understood byreading the subsequent detailed description and examples with referencesmade to the accompanying drawings, wherein:

[0020]FIG. 1a shows a cross-section of the biosensor of an embodiment ofthe invention.

[0021]FIG. 1b shows a cross-section of the sensing structure before thedeposition of urease, of an embodiment of the invention.

[0022]FIG. 1c shows a package of the sensing structure of an embodimentof the invention.

[0023]FIG. 2 shows the structure of the urea biosensor measurementsystem of an embodiment of the invention.

[0024]FIG. 3a shows the I_(DS) versus V_(G) curves of the sensitive filmat the different pH values in an embodiment of the invention.

[0025]FIG. 3b shows the V_(G) versus pH curve of the sensitive film atthe different pH values in an embodiment of the invention.

[0026]FIGS. 4a-4 e shows the voltage variation with respect to ureasolutions having concentrations of 1.25, 10, 40, 80, and 120 mg/dl,respectively, of the urea biosensor of an embodiment of the invention.

[0027]FIG. 5 shows the correcting curve between 1.25 mg/dl and 120 mg/dlurea solutions of urea biosensor.

DETAILED DESCRIPTION

[0028] The biosensor according to the invention has an extended gatefield effect transistor structure. The sensing film extended from thegate of the ISFET such that the metal oxide semiconductor field effecttransistor components can be separated from a tested solution to avoidthe instability of the semiconductor and the signal interference fromthe solution. In an embodiment of the invention, the urease isimmobilized by means of gel entrapment and forms a component of asensing film. The sensing film can be used to determine theconcentration of hydrogen ion or urea in a solution.

[0029] The biosensor according to an embodiment of the invention isillustrated in FIGS. 1a-1 c. Referring to FIG. 1 a, the biosensor 110having an extended gate field effect transistor structure comprises ametal oxide semiconductor field effect transistor, a sensing unit, and aconductive wire.

[0030] The metal oxide semiconductor field effect transistor 112 (shownas an electric circuit) is on a semiconductor substrate (not shown)which may be N- or P-type.

[0031] The sensing unit comprises a substrate 101, a silicon dioxidelayer 102, a tin oxide layer 103, and a urease layer 109. The substratecan be a conductive glass, such as an indium tin oxide (ITO) glass. Thesilicon dioxide is formed on the substrate with a thickness of 1000 Å to2000 Å and may function as a buffer allowing deposition of tin oxide onthe glass. Tin oxide layer has a thickness of 2000 Å to 3000 Å and isformed on the silicon dioxide layer. The substrate, the silicon dioxidelayer, and the tin oxide layer may available from a commercial product,such as SnO₂/SiO₂/glass, Kuanghua Development technical corporation,Taiwan, under the trademark TO-3030, for facilitating manufacture. Theurease layer is formed by photopolymerization of a mixture of aphotosensitive polymer and urease in a phosphate buffer solution. Thephotosensitive polymer and the urease are in a ratio ranging from 30:1to 5:1, preferably 25:1 to 15:1, and more preferably about 20:1 byweight. Furthermore, an insulating layer, for example, epoxy resin, maybe formed on the surface of the sensing unit to seal the sensing devicebut expose part of the urease layer for the contact with the testedsolution (not shown) and part of the conductive wire for electricalconnect.

[0032] The conductive wire 105 connects the metal oxide semiconductorfield effect transistor 112 and the sensing unit 110. The wire materialis the metal, such as, aluminum.

[0033] Briefly, the measurement of ion concentration of an acid or basesolution is attained by transforming the charge of hydrogen ionsadsorbed on the sensing film of the extended gate field effecttransistor into electrical signals, using the signals to control thewidth of the channel of MOSFET, and then determining the concentrationof the tested hydrogen ions by the magnitude of the electric current.

[0034] In the determination of the urea concentration of a solution,when urea reacts with the urease on the sensing film, OH⁻ or H⁺ ions areproduced through hydrolysis, the EGFET will change voltage correspondingto the pH value of the solution, and thus the urea concentration can beknown from the electric signals produced by the ISFET. The hydrolysis ofurea is as follows:

[0035] In the method of manufacturing a sensing unit according to anembodiment of the invention, the steps of providing a conductivesubstrate, forming a silicon dioxide layer on the conductive substrate,and forming a tin oxide layer on the silicon dioxide layer can beaccomplished by simply providing a commercially availableSnO₂/SiO₂/glass 106 product to simplify manufacturing and save time andreduce cost, or accomplished step by step as desired. The tin oxidelayer can be deposited on the silicon dioxide layer by chemical vapordeposition (CVD). The deposition temperature may be 250 to 600° C.,preferably 580 to 600° C. The layer has a thickness of preferably 0.2 to0.3 μm and a resistance of 20 to 30 Ω/□. For example, tin tetrachlorideand water are used to produce tin oxide layer. The reaction is shown asfollows:

SnCl₄+2H₂O→SnO₂+4HCl

[0036] The silicon dioxide layer can be deposited on the substrate byCVD at a temperature of preferably 580 to 600° C. The thickness ispreferably 100 to 300Å.

[0037] Referring to FIG. 1b, after the SnO₂/SiO₂/substrate is preparedor obtained, the tin oxide layer is connected to the conductive wire105. The surface of the resultant is preferably sealed with aninsulating layer 104, while part of the tin oxide layer is exposed forcoating urease as a detecting window and part of the conductive wire isexposed for connecting the gate of the metal oxide semiconductor fieldeffect transistor.

[0038] Next, a urease layer is immobilized on the exposed part of thetin oxide layer by gel entrapment. The gel entrapment is performed bymixing the photosensitive polymer and the urease in a phosphate buffersolution, photopolymerizing the resulting mixture, and then placing theresultant in the dark at a low temperature for a proper time, therebythe urease is immobilized on the tin oxide layer. The photosensitivepolymer may be, for example, a polyvinyl alcohol, which may have astyrylpyridinium group, such as PVA-SbQ. The photosensitive polymer andthe urease are used in a ratio ranging from preferably 30:1 to 5:1, morepreferably 25:1 to 15:1, and most preferably about 20:1 by weight. Forexample, the ratio may be in the range of 300 mg/100 μl PBS:10 mg/100 μlPBS to 50 mg/100 μl PBS:10 mg/100 μl PBS, preferably 250 mg/100 μlPBS:10 mg/100 μl PBS to 150 mg/100 μl PBS:10 mg/100 μl PBS. In thephotopolymerization, urease (in 5 mM phosphate buffer solution, pH 7)and photosensitive polymer (in 5 mM phosphate buffer solution) areradiated to react. The radiation may be UV light, such as, a UV lightwith a wavelength of 365 nm. After the photopolymerization, the resultmay be placed in the dark, for example, a dark box, at a low temperatureof, for example, 4° C. to −10° C., for a proper time, and thus theimmobilization of urease on the tin oxide layer, as well as the sensingunit, is accomplished, as shown in FIG. 1c. Note that, in the gelentrapment, white light should be avoided to prevent selfphotopolymerization of the enzyme.

[0039] The sensing unit can be placed directly in the tested solutionfor the determination of pH value or urea concentration.

[0040] Referring to FIG. 2, the biosensor 110 described above is used toconstruct the measuring system according to an embodiment of theinvention. The measuring system further comprises a reference electrode204, an instrumentation amplifier 202, a high-resistance multimeter 203,and a computer 205.

[0041] The reference electrode 204, for example, Ag/AgCl referenceelectrode, is immersed in the tested solution to help to maintain astable voltage and provide the function of calibration.

[0042] The instrumentation amplifier 202 amplifies the electric signalsand comprises two inputs and one output. The two inputs are connected tothe biosensor 110 and the reference electrode 204 through the conductivewires 108 and 206. The instrumentation amplifier may be, for example, acommercially available IC, LT1167. The connection of the biosensor 110and the instrumentation amplifier 202 can be accomplished by, forexample, pin connection, and therefore, after measurement, the biosensor110 and the instrumentation amplifier 202 are detachable toadvantageously renew the biosensor 110.

[0043] The high-resistance multimeter 203 is connected to the output ofthe instrumentation amplifier 202 to read out the output voltage fromthe biosensor 110.

[0044] The computer 205 is connected with the high-resistance multimeter203 through a communication interface card to store, process, or analyzedata. The computer 205 may be, for example, a personal computer. Thecommunication interface card may be, for example, HP82350. The parametermeasurement and the data storage may be controlled using HP VEE programinstalled in the computer. Microsoft Origin 6.0 is further used toanalyze output signals or plot graphs.

[0045] In the measurement, the urea sensing film 109 of the biosensor110 and the reference electrode are immersed in the tested solution 201containing acid, base, or urea.

EXAMPLES Example 1 Manufacturing the Biosensor

[0046] A commercial SnO₂/SiO₂/glass sold by Kuanghua Developmenttechnical corporation, Taiwan under the trade mark TO-3030 was cut intosquares of 1 cm×1 cm and washed in deionized water of an ultrasonicoscillator. An aluminum conductive wire was bonded to theSnO₂/SiO₂/glass 106 by silver glue and dried at 120° C. in an oven for10 minutes, and then cooled to room temperature. The aluminum conductivewire 108 was installed through a capillary 107 and the SnO₂/SiO₂/glassand the capillary were fixed by epoxy resin forming an insulating layer104 and dried at 120° C. in an oven for 20 minutes. The SnO₂/SiO₂/glasswas then packaged with epoxy resin but an area of 1.5 mm×1.5 mm was keptto be a sensing window. Then, one end of the aluminum conductive wirewas connected to a MOFET.

[0047] Subsequently, the sensing window was cleaned in deionized waterof the ultrasonic oscillator. A mixture of 200 mg PVA-SbQ (under trademark Toyo Gose sold by Kogyo Company, Japan) and 10 mg urease (EC3.5.1.5, powder, Type IV, from Jack bean, 50000 to 100000 units/g, soldby Sigma Chemical Company) in 200 μl phosphate buffer solution (5 mM, pH7.0) was prepared. 1 μl of the urease mixture was deposited on thesensing window. The resulting device was exposed under UV light (4 W/365nm) for photopolymerization for 20 minutes, then placed in a dark box at4° C. for about 12 hours, obtaining a EGFET with urea sensing film, asshown in FIG. 1c.

Example 2 Constructing Measuring System

[0048] Referring to FIG. 2, the biosensor having a urease film as asensing film as obtained from Example 1 was used. The sensing film ofthe biosensor was connected to the input of LT1167, and the output ofLT1167 was connected to a digital multimeter. An Ag/AgCl referenceelectrode was connected to another input of LT1167. A computer wasconnected with the digital multimeter through a communication interfacecard HP82350. The parameter measurement and the data storage wereperformed using HP VEE program installed in the computer. MicrosoftOrigin 6.0 was used to process and analyze output signals.

[0049] A series of solutions having pH value of 1, 3, 5, 7, and 9 weremeasured using the measuring system as described above. By changing gatevoltage, output electric currents were obtained. The data were processedand analyzed using Microsoft Origin 6.0 to plot a curve of electriccurrent versus gate voltage, as shown in FIG. 3a. As the proper currentvalue, I_(DS), was about 300 μA, a high linear sensitivity for measuringa solution having a pH value in the range of 1 to 9 was obtained to be58 mV/pH. A curve of gate voltage versus pH value was also plotted, asshown in FIG. 3b.

[0050] A series of urea solutions having concentrations of 1.25 mg/dl,10 mg/dl, 40 mg/dl, 80 mg/dl, and 120 mg/dl were measured using themeasuring system as described above. Curves of output voltage versusmeasuring time were plotted as shown in FIGS. 4a to 4 e.

[0051] After linear calibration of the data obtained above, thelinearity of the sensor was obtained in the range of 5 mg/dl to 50mg/dl, as shown in FIG. 5 showing the plot of voltage difference versusurea concentration.

[0052] The results indicate that the sensor and the manufacturing methodof the sensing unit of an embodiment of the invention have advantages oflow cost, easy attainment, and a simple package due to the utilizationof commercially available SnO₂/SiO₂/glass. The method according to theinvention is novel.

[0053] Furthermore, a precise response for the biosensor to solutionshaving different pH values or urea concentrations can be obtained by themeasuring system according to an embodiment of the invention, and inturn the pH value or the urea concentration can be obtained precisely.Furthermore, the sensor and the readout circuit are detachable and thesensing unit is disposable, thus, can be prevented contamination ordamage to the sensing film.

[0054] While the invention has been described by way of example and interms of the preferred embodiments, it is to be understood that theinvention is not limited to the disclosed embodiments. To the contrary,it is intended to cover various modifications and similar arrangements(as would be apparent to those skilled in the art). Therefore, the scopeof the appended claims should be accorded the broadest interpretation soas to encompass all such modifications and similar arrangements.

What is claimed is:
 1. A biosensor having an extended gate field effecttransistor structure, comprising: a metal oxide semiconductor fieldeffect transistor on a semiconductor substrate; a sensing unitcomprising a substrate, a silicon dioxide layer on the substrate, a tinoxide layer on the silicon dioxide layer, and a urease layer immobilizedon the tin oxide layer; and a conductive wire connecting the MOSFET andthe sensing unit.
 2. The biosensor as claimed in claim 1, wherein themetal oxide semiconductor field effect transistor is an N-type fieldeffect transistor.
 3. The biosensor as claimed in claim 1, wherein theconductive wire comprises aluminum.
 4. The biosensor as claimed in claim1, wherein the substrate is the conductive substrate.
 5. The biosensoras claimed in claim 4, wherein the substrate is an indium tin oxideglass.
 6. The biosensor as claimed in claim 1, wherein the urease layeris immobilized on the tin oxide layer by gel entrapment.
 7. Thebiosensor as claimed in claim 6, wherein the urease layer is formed byphotopolymerization of a mixture of a photosensitive polymer and ureasein a phosphate buffer.
 8. The biosensor as claimed in claim 7, whereinthe photosensitive polymer and the urease are in a ratio ranging from30:1 to 5:1 by weight.
 9. The biosensor as claimed in claim 1, furthercomprising an insulating layer on the surface of the sensing unit butexposing part of the urease layer and part of the conductive wire. 10.The biosensor as claimed in claim 9, wherein the insulating layercomprises epoxy resin.
 11. The biosensor as claimed in claim 1, whereinthe substrate, the silicon dioxide layer, and the tin oxide layer areformed as a SnO₂/SiO₂/glass sold by Kuanghua Development TechnicalCorporation, Taiwan under the trademark TO-3030.
 12. A method ofmanufacturing a sensing unit, comprising: providing a conductivesubstrate; forming a silicon dioxide layer on the conductive substrate;forming a tin oxide layer on the silicon dioxide layer; electricallyconnecting the conductive substrate with a conductive wire; forming aninsulating layer on the surface of the sensing unit but exposing part ofthe tin oxide layer and part of the conductive wire; and immobilizing aurease layer on the exposed part of tin oxide layer by gel entrapment.13. The method as claimed in claim 12, wherein the gel entrapment isperformed by mixing the photosensitive polymer and the urease in aphosphate buffer solution, photopolymerizing the resulting mixture, thenplacing the result in the dark at a low temperature for a proper time,thereby the urease is immobilized on the tin oxide layer.
 14. The methodas claimed in claim 13, wherein the photosensitive polymer is apolyvinyl alcohol.
 15. The method as claimed in claim 14, wherein thepolyvinyl alcohol has styrylpyridinium groups.
 16. The method as claimedin claim 13, wherein the photosensitive polymer and the urease are in aratio ranging from 30:1 to 5:1 by weight.
 17. The method as claimed inclaim 13, wherein the urease is used in a form of solution in a 5 mMphosphate buffer solution in a concentration of 10 mg/100 μl with a pHvalue of 7, the photosensitive polymer is used in a form of solution ina 5 mM phosphate buffer solution in a concentration of 200 mg/100 μlwith a pH value of
 7. 18. The method as claimed in claim 13, wherein thephotopolymerization is performed with a UV light having a wavelength of365 nm.
 19. The method as claimed in claim 13, wherein the lowtemperature is 4° C.
 20. The method as claimed in claim 12, wherein thesubstrate, the silicon dioxide layer, and the tin oxide layer areprovided as a SnO₂/SiO₂/glass sold by Kuanghua Development TechnicalCorporation, Taiwan under the trade mark TO-3030.
 21. A measuringsystem, comprising: a biosensor as claimed in claim 1; a referenceelectrode for a stable voltage; an instrumentation amplifier having twoinputs and one output, wherein the two inputs are connected with thebiosensor and the reference electrode, respectively; a high-resistancemultimeter connected with the output of the instrumentation amplifier;and a computer connected with the high-resistance multimeter through acommunication interface card to store, process, or analyze data.
 22. Themeasuring system as claimed in claim 21, wherein the instrumentationamplifier is LT1167.
 23. The measuring system as claimed in claim 21,wherein the reference electrode is an Ag/AgCl reference elctrode. 24.The measuring system as claimed in claim 21, wherein the computer is apersonal computer.
 25. The measuring system as claimed in claim 21,wherein the communication interface card is HP82350.
 26. The measuringsystem as claimed in claim 21, wherein HP VEE program is further used tocontrol the parameter measurement and the data storage.
 27. Themeasuring system as claimed in claim 26, wherein Microsoft Origin 6.0 isfurther used to analyze output signals or plot graphs.